7-(2-Aminomethylphenyl-acetamido)-3-{8 2-(5-hydroxymethyl-1,3,4-thiadiazolyl)-thiomethyl{9 -3-cephem-4-carboxylic acid

ABSTRACT

7-(2-Aminomethylphenylacetamido-3-(heterocyclicsubstituted)thiomethyl-3 -cephem-4-carboxylic acids in which the heterocyclic substituent is 2-(1,3,4-thiadiazolyl)- or 2-(5hydroxymethyl-1,3,4-thiadiazolyl)-, and their nontoxic, pharmaceutically acceptable salts and their Schiff bases, as made by reaction of salicylaldehyde with the free amino group, are valuable as antibacterial agents and are particularly valuable as therapeutic agents in poultry and animals, including man, in the treatment of infectious diseases caused by many Gram-positive and Gram-negative bacteria.

United States Patent H 1 nu 3,910,899

Gottstcin et al. Oct. 7, 1975 7-(2-AMINOMETHYLPHENYL-ACETAMIDO)-3-[2-(5-HY[)ROXYMETHYL- Primary ExaminerNich0las S. Rizzo1,3,4.TH]A Z() 1.3- Attorney, Agent, or FirmHerbert W. Taylor, Jr.CEPHEMA-CARBOXYLIC ACID [75] Inventors: William J. Gottstein,Fayetteville;

Murray A. Kaplan, Syracuse; [57] ABSTRACT Alphonse P. Granatek,Baidwinsville, of 7-(Z-Aminomethylphenylacetamid0-3'(heterocyclic- [73]Assignee: BrismbMyers Company, New Yorksubstituted)thiomethyl-3-cephem-4-carb0xylic acids in which theheterocyclic substituent is 2-(1,3,4- thiadiazolyl or2(5-hydr0xymethyl-l ,3,4- Filed? sept- 1 1973 thiadiaz0lyl)-, and theirnontoxic, pharmaceutically [21] Appll No; 395,102 acceptable salts andtheir Schiff bases, as made by reaction of salicylaldehyde with the freeamino group, are valuable as antibacterial agents and are particu- Cl260/243 424/245; 260/240 0 larly valuable as therapeutic agents inpoultry and ani- [51] Int. Cl. C07D 501/22 mals, induding man, in thetreatment f i f ti 0 Search t v C eases aused many c an ppgsitive andGram.

negative bacteria. [56] References Cited UNITED STATES PATENTS 5 Claims,No Drawings 3,766,175 lO/l973 Lemieux el al. 260/243 C7-(Z-AMINOMETHYLPHENYL-ACETAMIDO)-3- [Z-(S-HYDROXYMETHYL-l,3,4-THIADIAZOLYL)- TH]OMETHYLl-3-CEPHEM-4-CARBOXYLIC ACID BACKGROUND OF THEINVENTION 1. Field of the lnvention The cephalosporins of the presentinvention possess the usual attributes of such compounds and areparticularly useful in the treatment of bacterial infections byparenteral administration.

2. Description of the Prior Art The cephalosporins are a well-knowngroup of semisynethtic antibacterial agents made originally, forexample, by acylation of the 7-amino group of the nucleus7-aminocephalosporanic acid (7-ACA) and later by similar acylation ofnuclei derived therefrom, as by modification of its substituent at the3-position. Various reviews have appeared in the scientific literature(e.g. Cephalosporins and Penicillins Chemistry and Biology, edited byEdwin H. Flynn, Academic Press, New York, 1972, and particularly pages554-569) and in the patent literature, e.g. as in US. Pat. Nos.3,687,948 and 3,741,965 (both US. Class 260-243C).

Recently issued patents on 3-thiolated cephalosporins in which the7-substituent is a. a-Amino-a-phenylacetamido include US. Pat. Nos.3,641,021, 3,734,907, 3,687,948, 3,741,965, Japan 7124400 (Farmdoc463748), Belgium Pat. No. 776,222 (Farmdoc 389831), Belgium Pat. No.772,592 (Farmdoc 196961), West Germany Pat. No. 2,202,274 (Farmdoc50428T) corresponding to US. Ser. No. 167,534 filed July 29, 1971Netherlands Pat. No. 7205644 (Farmdoc 76309T); and

b. o-, mor p-aminoethoxyphenylacetamido as Netherlands 72/13968 (Farmdoc24740U) corresponding to US. Ser. No. 189,369 filed Oct. 14, 1971; and

c. o-aminomethylphcnylacetamido as Netherlands 72/06326 (Farmdoc76374'1) (which also reviews the older patent literature concerningsubstituted 7- phenylacetamidocephalosporanic acids) corresponding toU.S. Ser. No. 142,337 filed May 11, 1971; and

d. N-(phenylacetimidoyl)aminoacetamido as US. Pat. No. 3,692,779; and

e. a-amino-a-(1,4-cyclohexadienyl)acetamido as in Belgium 776,222(Farmdoc 38983T).

Additional similar disclosures are found in US. Pat. No. 3,692,779(Belgium Pat. Nov 771,189; Farmdoc 12,817T), Japan Pat. No. 72/0555()(Farmdoc 12,921T), Japan 72/05551 (Farmdoc 12,922T), Japan Pat. No.7l/24400(Farmdoc 46,374T), Belgium Pat.

No. 776,222 (Farmdoc 38,983T).

Recently issued patents containing a partially hydrogenated benzene ringin the 7-substituent but lacking a thiomethyl group at the 3-positioninclude those in which the 7-substituent is a. 2-( l,4-cyclohexadien-l-yl)acetamido as in US. Pat. No. 3,704,297 (Farmdoc78154T) and Belgium Pat. No. 759,326 (Farmdoc 38,1725); and

b. a-amino-a-( l ,4-cyclohcxadienyl )acetamido and related compounds asin Us Patv No. 3,485,819; West Germany 2,152,745 (Farmdoc 296061"); and

c. a-amino-a-( 1,2-cyclohexeny1 )acetamido as in Belgium 773,773(Farmdoc 25515T); and

d). p-(a-aminoalkyl)phenylacetamido as in US. Pat. No. 3,382,241; and

e. o-aminomethylphenylthioacetamido as in US. Pat. No. 3,657,232.

SUMMARY OF THE INVENTION This invention comprises the compounds of theformula and their nontoxic, pharmaceutically acceptable salts and Schiffbases, as with salicylaldchyde.

Such salts include carboxylic acid salts including nontoxic metallicsalts such as sodium, potassium, calcium and aluminum, the ammonium saltand substituted ammonium salts, e.g., salts of such nontoxic amines astrialkylamincs, including triethylamine, pro caine, dibenzylamine,N-benzylbeta-phenethylamine, 1-ephenamine, N,N'-dibenzy1ethylenediamine,dehydroabietylamine, N,N'-bisdehydroabietylcthylenediamine, N-(lower)-alkylpiperidine, e.g., N- ethylpiperidine, and other amines whichhave been used to form salts with benzylpenicillin; and in all cases thenontoxic, acid addition salts thereof i.e., the amine salts) includingthe mineral acid addition salts such as the hydrochloride, hydrobromide,hydroiodide, sulfate, sulfamate and phosphate and the organic acidaddition salts such as the maleate, acetate, citrate, oxalate,succinate, benzoate, tartrate, fumarate, malate, madelate, ascorbate andthe like.

The compounds of the present invention are prepared according to thepresent invention by coupling with a compound of the formula COOHwherein R has the meaning set out above (or a salt or easily hydrolyzedester thereof including those of US. Pat. No. 3,284,451 and UK. Pat. No.1,229,453 and any of the silyl esters described in US. Pat. No.3,249,622 for use with 7-aminopenicillanic acid and used in GreatBritain Pat. No. 1,073,530) a particular acid or its functionalequivalent as an acylating agent for a primary amino group. Aftercoupling, the blocking group is removed to give the desired product.Said acid has the formula wherein B represents a blocking group of thetype used either in peptide syntheses or in any of the numeroussyntheses of a-aminobenzylpenicillin from 2- phenylglycine. Particularlyvaluable blocking groups are a proton, as in the compound of the formulaor a B-diketone as in Great Britain Pat. No. l, 1 23,333, e.g., methylacetoacetate, in which case the acid containing the blocked amino groupis preferably converted to a mixed anhydride, as with ethylchloroformate, before reaction with compound ll or a salt thereof toform the desired product l after acid cleavage.

Further to the discussion above of blocking groups used on the freeamino group of the sidechain acid during its coupling with compound ll,the blocking group is then removed to form the products of the presentinvention, e.g., the t-butyoxy-carbonyl group is removed by treatmentwith formic acid, the carbobenzyloxy group is removed by catalytichydrogenation, the 2-hydroxy-l-naphthcarbonyl group is removed by acidhydrolysis and the trichloroethoxycarbonyl group by treatment with zincdust in glacial acetic acid. Obviously. other functionally equivalentblocking groups of an amino group can be used and subh groups areconsidered within the scope of this invention.

Thus, with respect to said acid to be used to couple with compound II,functional equivalents include the corresponding acid anhydridcs,including mixed anhydridcs and particularly the mixed anhydridesprepared from stronger acids such as the lower aliphatic monoesters ofcarbonic acid, or alkyl and aryl sulfonic acids and of more hinderedacids such as diphenylacetic acid. ln addition, an acid azide or anactive ester of thioestcr (e.g., with p-nitrophenol, 2,4-dinitrophenol.thiophenol, thioacetic acid) may be used or the free acid itself may becoupled with compound I] after first reacting said free acid withN,N'-dimethylchloroformiminium chloride [cf. Great Britain, l,()()8,l70and 4 Novak and Weichet, Experientia XXL 6, 360 (1965)] or by the use ofenzymes or of an N,N'- carbonyldiimidazole or an N,N'-carbonylditriazole[cf. South African patent specification 63/2684] or a carbodiimidereagent [especially N,N'-dicyclohexylcarbodiimidc,N,N'-diisopropylcarbodiimide or N- cyclohexyl-N'-( 2-morpholinoethyl)carbodiimide; cf. Sheehan and Hess, J. Amer. Chem. Soc., 77, I067(1955)] or of alkylamine reagent [cf. R. Buijle and HG. Viehe, AngewChem. lntemational Edition 3, 582

( 1964)], or ofa ketenimine reagent ]cf. C. L. Stevens and M.E. Mond, J.Amer. Chem. Soc., 80, (4065)] or of an isoxazolium salt reagent ]cf. R.B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc. 83, 1010l96l )1. Another equivalent of the acid chloride is a correspondingazolide, i.e., an amide of the corresponding acid whose amide nitrogenis a member or an quasiaromatic five-membered ring containing at leasttwo nitrogen atoms, i.e., imidazole, pyrazole, the triazoles,benzimidazole, benzotriazole and their substituted derivatives. As anexample of the general method for the preparation of an azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolarproportions at room temperature in tetrahydrofuran, chloroform,dimethylformamide or a similar inert solvent to form the carboxylic acidimidazolide in practically quantitative yield with liberation of carbondioxide and one mole of imidazole. Dicarboxylic acids yielddimidazolide. The by-product, imidazole, precipitates and may beseparated and the imidazolide isolated, but this is not essential. Themethods for carrying out these reactions to produce a cephalosporin andthe methods used to isolate the cephelosporin so produced are well knownin the art.

In the treatment of bacterial infections in man, the compounds of thisinvention are administered parenterally, in accordance with conventionalprocedures for antibiotic administration, in an amount of from about 5to 200 mg./kg./day and preferably about 5 to 20 mg./kg./day in divideddosage, e.g., three to four times a day. They are administered in dosageunits containing, for example, or 250 or 500 mg. of active ingredientwith suitable physiologically acceptable carriers or excipients. Thedosage units are in the form of liquid preparations such as solutions orsuspensions.

The present invention provides for the production of the new compoundstherein by the processes of the following reaction scheme:

com

:CH,COOH

N N- R :is or S CIMOH Certain 3-substituted 7-[ a-( 2- HOaminomethylphenyl)acetamidolcephalosporanic acid derivatives (A; seeNetherlands 72/06326, Farmdoc Q 763741, corresponding to U.S. Ser. No.l42,337 filed May ll, 1971) provide a series of parenteral-use ca- 5 5phalosporins which are very effective derivatives with a broad spectrumof activity. Their limited water i' M Z solubility 2 mg./ml.) has,however, caused crystal C luria formation even when the antibiotics hasbeen 7 administered parenterally as a readily-dissociable O solublederivative. The object of the present invention dOOH was, therefore, toobtain equally active compounds which show higher water solubility asthe zwitterion form and do not cause crystalluria.

or a nontoxic, pharmaceutically acceptable salt wherein R is i thereof.(H CO N i T The solubilities were determined in (Ll M pH 7.0 phosphatebuffer.

especially wherein R is COOH N N N R Solubility l I 3U N--N L9 m ./ml.(1 N /LCH .1 g

'T (MR-S94) or s (MR S96 L CH" IG N N (1.9 mg/ml, (2) The objective ofthe present invention was achieved lL -CH; by the provision, accordingto the present invention of 135 mg/mL an acid having the formula &5(BL-S685) N- 2.23 mg./ml. 40 ll I L H N H. /-CH,0H

(BLSOQU) s CHu(v NH CH CH/ I. This compound (also called MR-S94 isclaimed in l U.S. Ser. No. l42,337 filed May I l, I971 (see Netherlands72/06326; Fanndoc 76,3741). 0 C 2. This compound (also called MR-S96) isclaimed in U.S. Ser. No. l42,377 filed May I l, l97l (see Netherlands72/06326; Farmdoc 76,374T).

STARTING MATERIALS Methyl o-bromomethylphenylacetate A mixture of methylo-methylphenylacetate (82.0 g.,

0.50 mole), N-bromosuccinimide (89.0 g., 0.50 mole),

o-Azidomethylphenylacetic acid A mixture of methylo-bromomethylphenylacetate (90.1 g., 0.371 mole), sodium azide (26.0 g.,0.40 mole) and 10% aqueous acetone (750 ml.) was stirred at roomtemperature for 3 h. The solvent was removed under reduced pressure andthe residue treated with ether (300 ml.) and water 100 ml. The crudemethyl o-azidomethylphenylacetate (74.8 g.), obtained after drying andconcentrating the ether solution, was dissolved in 150 ml. of methanol.This solution was cooled in ice and treated with 150 ml. 3 N methanolicsodium hydroxide. The mixture was left at room temperature for l h.,then concentrated to dryness and the residue dissolved in water. Theaqueous solution was acidified, the product collected by filtration,dried and recrystallized from ethyl acetate-n-hexane to give 49.5 g.(70%) of the acid, m.p. 1 16-l 18; n.m.r. (CDCl sharp singlets at 72.75(4H), 5.63 (2H) and 6.28 (2H); 'y,,,,, 2100 and 1700 cm.

Anal. Calcd. for C H N O C, 56.53; H, 4.75; N, 21.98.

Found: C, 56.37; H, 4.65; N, 21.74

o-Aminomethylphenylacetic acid A mixture of o-azidomethylphenylaceticacid (9.6 g., 0.050 mole), 10% Pd on charcoal (2.5 g.), methanol 150ml.) and l N hydrochloric acid (50 ml.) was bydrogenated at 30 psi. for3.5 h. The mixture was filtered, concentrated under reduced pressure toa volume of approximately 30 ml. and extracted with ether. From theether extract 1-2 g. of impure starting material was recovered. Theaqueous solution was adjusted to pH 5.0 with dilute ammonium hydroxideand cooled in ice. The white solid precipitate was collected by filtration, washed successively with ice-water, methanol and ether, anddried in vacuo over P Yield 5.4 g. (65%), mp. 179-181(decomp.);n.m.r.(CF CO H 'r 2.54 (s, 4H) 5.48 (q, 2H) and 6.00 (s, 2H).

o-tert-Butoxycarbonylaminomethylphenylacetic acid Triethylamine 14.4 g.,0. 143 mole) was added to an ice-cooled suspension ofo-aminomethylphenylacetic acid (10.3 g., 0.0624 mole) in 100 ml. ofwater followed by the addition of a solution of tertbutoxycarbonyl azide(l 1.4 g., 0.080 mole) in ml. of THF. The reaction mixture was stirredat room temperature for 16 h., then most of the THF was removed underreduced pressure. The aqueous solution was washed with ether, layeredwith 125 ml. of ethyl acetate and with ice-cooling brought to pH 3.5with dilute hydrochloric acid. The ethyl acetate solution was dried,concentrated and the solid residue recrystallized from ethylacetate-n-hexane (1:1) to give 14.1 g. (87%) of white needles mp. l l4-l16.

Anal. Calcd. for C H NO C, 63.39; H, 7.22; N, 5.28.

Found: C, 63.44; H, 7.21; N, 5.42.

2 ,4-Dinitrophenyl o-tert.-butoxycarbonylaminophenylacetateN,N'-Dicyclohexylcarbodiimide (1.0 g., 0.0050 mole) was added to anice-cooled solution of o-tertbutoxycarbonylaminomethylphenylacetic acid(1.33 g., 0.0050 mole) and 2,4-dinitrophenol (0.92 g., 0.0050 mole) in12 ml. of anhydrous tetrahydrofuran. The reaction mixture was kept atroom temperature for one hour, then the precipitatedN,N'-dicyclohexylurea was removed by filtration. The solvent was removedfrom the filtrate to give the activated ester as a viscous yellow oil.

o-tert.-Butoxycarbonoylaminomethylphenylacetic acid can be prepared inquantitative yield from tert.- butoxycarbonyl azide and the amino acidby using triethylamine as the base.

The BOC-amino acid reacts with thionyl chloride in the presence oftriethylamine (methylene chloride as solvent) or pyridine (benzene assolvent) to give the BOC-amino acyl chloride which can be directlycoupled with the compound of the formula 5 ut lCH,S-R

c op mNH ("H. .NH PCIS 1. HO =NOH E heat CHCL, =0 below 0C. 2. NH,OH LHCOOH J. Org. Chem. 9. 380-391 1944) and 28, 2797-28114 1 1963 MaterialsWL, g. Vol, ml. Moles Z-lndunone oxime 1000 6.78 Phosphoruspentachlnride 1482 7.13 (hlurolhrm 56600 Sodium Hydroxide Solution 6X0Dark-u KB" activated charcoal Procedure 1. Dissolve 1000 g. of2-indanone oxime in 31600 ml. of chloroform at 2025 C.

2. Cool the solution to 30 C. [On cooling the 2- indanone oxime solutionto 30 C., some of the oxime crystallizes.

3. Add 1482 g. of phosphorus pentachloride to the vigorously stirredsuspension in portions. Control the temperature of the reaction at 28 to32 C. by the rate of addition of the solid phosphorus pentachloride.[The best results were obtained on running the reaction at 30 C. It canalso be successfully run at l to -5 C. or perhaps even higher but itappears that more tar and side products are produced which thencomplicate isolating the lactam.]

4. Stir the reaction at -30 C. for minutes after completing the additionand then warm it to 25 C. over three-fourths hr. During this period thesolids dissolve and then a new solid reprecipitates.

5. Stir the reaction at 25 C. for 3 additional hours and then withthorough mixing add it to 31600 ml. of water at 05 C. [The reaction canbe followed by TLC (thin layer chromatography). In the system of 8 partsbenzene and 2 parts acetic acid the lactam has an Rf 0.36 and the oximehas as Rf= 0.64. The spots are developed by .05% potassium permanganatespray. The oxime spot may not completely disappear but it should becomequite faint] Subsequent washing operations were conducted at 25 C.

6. Separate the layers and wash the chloroform phase with 15,800 ml. ofwater.

7. Combine the water fractions and extract them with 15,800 ml. ofchloroform.

8. Combine the chloroform fractions, layer with 15,800 ml. of water andwith good mixing titrate the mixture to pH 7 with 10% sodium hydroxidesolution. This may take about 680 ml. of sodium hydroxide solution andthe titration is slow. [This basic wash is important in removing the tarproducing side products. The titration may take 1 to 2 hours.)

9. Separate the layers and wash the chloroform with 15,800 ml. of water.

Combine the water fractions, wash with 9200 ml. of chloroform andcombine the chloroform fractions.

1 1. Carbon treat the chloroform solution with 1000 g. of activatedcharcoal (Darko KB") at for 15-30 min.

12. Filter the slurry through diatomaceous earth (Dicalite) wash thecake with chloroform and concentrate the filtrate at reduced pressure toleave o-aminomethylphenylacetie acid lactam as a dry solid.

13. The yield of crude lactam is nearly 100%. It is a yellow crystallinesolid. [If the tar making materials have not been removed by the washes,this produce will come out dark. It can be recrystallized from hot waterafter first adjusting the water slurry to pH 7.0 or fromtoluene-heptane.

Procedure 1. Add 8000 ml. of concentrated hydrochloric acid to theapproximately 1,000 g of crude lactam obtained from oxime rearrangement.

2. Stir the mixture and heat it cautiously to reflux for 3 hours. [Onheating this reaction an excessive amount of foam is formed as excessHCI leaves. This foam can fill the whole apparatus. It can be reduced bysilicon antifoam agent. After the initial foam stage is passed, thereaction can be refluxed without difficulty] 3. Cool the dark slurry toabout 40-50 C. and add g. of activated charcoal (Darko KB") and continuestirring.

4. Carbon treat for 15-20 min. filter the slurry through a Dicalite cakeand wash the cake with about 4000 ml. of hot water.

5. Extract the clear yellow filtrate with 6000 ml. of methylene chloride(one-half volume) and separate the methylene chloride. Save the CH CIlayer for checking possible recovery of unchanged lactam.

6. Concentrate the water phase at reduced pressure to give solido-aminomethylphenylacetic acid hydrochloride.

7. Add MlBK to the wet solids and continue the reduced pressuredistillation and MIBK addition until all the water has been removed fromthe solids.

8. Continue the reduced pressure distillation until all the MlBK hasbeen removed from the solids. [The MlBK distillation not only azeotropesthe water but also takes excess HCl with it.)

9. Redissolve the solids in 3900 ml. of water and add 650 ml. of MIBK.

10. While stirring at 2025 C. adjust the pH of the solution to 5.0 with6 N ammonium hydroxide. The o-aminomethylphenylacetic acid zwitterionstarts to crystallize at about pH 3.5.

l 1. Stir the zwitterion slurry and cool it to 05 C. for 1 hr.

12. Filter the slurry, wash the cake carefully with -l000 ml. of icecold water, then 2,000 ml. of MlBK and then 5,000 ml. of ice coldacetone. The combined filtrate and washes should be checked for lactamcontent.

l3. Suck the cake dry and then dry it in an air circulating oven at 45C. The yield is 670-730 g; 60-65% based on oxime.

14. The reactions and processing may be followed by TLC using thesolvent system 5 acetone, 1.5 benzene, 1.0 acetic acid, 1.5 water;developed by KMnO R lactam 0.88, R, amino acid 0.69.

o-Aminomethylphenylacetic acid hydrochloride In a 500 ml. round-bottomflask, fitted with a reflux condenser, and a magnetic stirrer. is placed10.1 g. (0.075 moles) of o-aminomethylphenylacetic acid lactam and 100m]. of concentrated hydrochloric acid. The mixture is then refluxed for3 hr. While hot. the reaction mixture is treated with 2.0 g. of charcoal(Darko KB) for 5 min., and filtered. The filtrate is concentrated todryness at 50-60/l 5mm and finally at high vacuum over P 0 (Note I Thesolid is recrystallized from a preformed mixture of acetonezwater, 15:1(Note 2). The hydrochloride is dried in a vacuum desiccator over P 0 Theyield of the pure product, melting at 188-l90, is 11.4 g. (78%).

Anal. Calcd. for C,,H, NO Cl: C, 53.73; H, 5.97; N, 6.96; Cl, 17.66.

Found: C, 53.56; H, 6.02; N, 6.89; Cl, 17.76.

Notes 5 1. For a successful recrystallization absolutely dry material isneeded.

2. The ratio of acetone to water may vary, depending on the dryness ofthe crude hydrochloride.

Potassium o-[ l-carbomethoxypropen-2-ylaminomethyl]- phenylacetate In a2000 ml. round-bottom flask, fitted with a reflux l5 condenser and anoverhead stirrer, was placed 45.92 g. (0.28 moles) ofo-aminomethylphenylacetic acid, 15.28 g. (0.28 moles) of KOH, 64.96 g.(0.56 moles) of methyl acetoacetate and 1500 ml. of absolute methanol.The mixture was then refluxed for 3 hr. The solution was thenconcentrated to a small volume 100- l 50 ml.), filtered and diluted with400-700 ml. of anhydrous ether. Upon scratching, the productcrystallized out. The solid was filtered and dried in a vacuumdesiccator over P 0 (The solid material was extremely hygroscopic. itshould be filtered as quickly as possible and should not be air-dried.)The yield of product, melting at 140148, was 84.] g. (99%).

Anal. calcd. for C H,.,NO,K: C, 54.20; H, 5.49; N, 4.52; KF, 2.90.

Found: C, 53.72; H, 5.44; N, 4.56; KF, 292

nylacetate To a ethanolic solution g. sodium ethoxide which was preparedfrom 0.6 g (0.026 atom) of metallic sodium and 50 ml. of absoluteethanol were added 4.27 g. (0.026 mole) of o-aminomethylphcnylaceticacid and 3.38 g. (0.026 mole) of ethyl acetoacetate and the mixture wasrefluxed for 6 hours. The mixture was evaporated to dryness and theresidue was recrystallized from ethanol to give 6.36 g. (82%) of sodiumo-[ lethoxycarbonyl-1-propen-2-ylaminomethyl]phenylacetate as colorlessneedles melting at 230232 C.

IR: 65 3320, 1645, 1605, 1470, 1395, 1275, l 180 cm.

Anal. calcd for C H No Naz C, 60.20; H, 6.06; N, 4.68.

Found: C, 59.95; H, 5.86; N, 4.67.

t-Butoxycarbonyl azide To a cooled solution of 100 g. (0.76 mole) oft-butyl carbazate in 87 g. of glacial acetic acid and 120 ml. of waterwas added dropwise a solution of 60 g. (0.85 mole) of sodium nitrite in50 ml. of water over a period of 40 minutes, the temperature being keptat l0l5 C. After the addition was completed the stirring was continuedfor an additional 30 min. at the same temperature. To the mixture wasadded 100 ml. of water and a separated oil was extracted with five 100ml. portions of methylene chloride. The combined organic extracts werewashed with 100 ml. of 10% sodium bicarbonate solution and 100 ml. ofwater successively, and dried over anhydrous sodium sulfate. Themethylene chloride was removed under diminished pressure on a water bathmaintained at 40-45 C. The residual azide was distilled and collected at45C./20 mm Hg. It weighed 92.7 g. (84%).

o-( t-Butoxycarbonylaminomethyl )phenylacctic acid To a solution of g.(0.35 mole) of o-aminomethylphenylacetie acid hydrochloride and l 16 g.1.15 moles) of triethylamine (TEA) in 400 ml. of water was addeddropwise a solution of 64 g. (0.45 mole) of tbutoxycarbonyl azide in 300ml. of tetrahydrofuran (THF) under stirring at 0 C. After the additionwas completed, the temperature was allowed to rise to room temperatureand the stirring was continued for 20 hours. The tetrahydrofuran wasdistilled of! below 40 C. and the aqueous solution was washed with 200ml. of ether, layered with 200 ml. of ethyl acetate and acidified withdil. hydrochloric acid to pH 3 under cooling at 0 C. The organic layerwas separated and the aqueous layer was extracted with four 200 ml.portions of ethyl acetate. The combined ethyl acetate solution waswashed with 200 ml. of water, dried over anhydrous sodium sulfate andconcentrated in vacuo. The concentrate was treated with 500 ml. ofn-hexane to give 87.9 g. of o-(t-butoxycarbonylaminomethyl )phenylaceticacid as colorless needles melting at l l4l 16 C.

2,4-Dinitropphenyl o-t-butox ycarbonylaminomethylphenylacetateDicyclohexylcarbodiimide 17.72 g., 0.086 mole) (DCC) was added in oneportion to a mixture of o-(tbutoxycarbonylaminomethyl)phenylacetic acid(22.73 g., 0.086 mole) and 2,4-dinitrophenol 15.82 g., 0.086 mole)(2,4-DNP) in 250 ml. of THF. The reaction mixture was stirred for 2hours at room temperature. The precipitated dicyclohexylurea wasfiltered off and washed with ml. of THF. The filtrate and washings werecombined and concentrated under reduced pressure below 50 to give aviscous yellow oil which was triturated with n-hexane ml.) to afford2,4- dinitrophenyl o-t-butoxycarhonylaminomethylphcnylacetic acid asyellow needles. Yield 34.9 g. (94% m.p. 76-77 C.

IR: 'y,,,,,,""" 3340, 1785, 1685, 1610, 1540, 1530, 1500, 1340 cm.

Anal. Calcd. for C ,,H ,N;,O,,: C, 55.68; H, 4.9]; N, 9.74.

Found: C, 55.70; H, 5.05; N, 9.93.

o-tert.-Butoxycarbonylaminomethylphenylacetic acid can be prepared inquantitative yield from tert.- butoxycarbonyl azide and the amino acidby using triethylamine as the base.

The BOC-amino acid reacts with thionyl chloride in the presence oftriethylamine (methylene chloride as solvent) or pyridine (benzene assolvent) to give the BOC-amino acyl chloride which can be directlycoupled with the compound of the formula wherein R has the meaning setout above in methylene chloride solution in the presence oftriethylamine. The protecting group can subsequently be removed bytreatment with cold trifluoroacetic acid.

7-Amino-3-[ 2-( 1,3,4-thiadiazoly1 )-thiomethyl ]-3- cephem-4-carboxy1icacid OOH 2-Mercapto-1,3,4-thiadiazole The procedure of .l. Goerdeler..1. Ohm and O. Tegtmeyer. Bericlite, 89, 1534 (1956) was followed. To asolution of 160 ml. of 48% hydrobromic acid and 100 mg. of powderedcopper at -7 was added slowly with alternation 13.6 g. (0.1mole)thiadiazole (Eastman) and 32 g. of sodium nitrate in small portions overa period of Va hour. The mixture was stirred for l /2 hours at and for 1hour at room temperature. The mixture was then neutralized with 50%potassium hydroxide to pH 9.5. The mixture was filtered and the filtratewas continuously extracted with ether for 6 hours. The ether wasevaporated to mm to a solid which was dissolved in 40 ml. of ethylalcohol and treated with 5 g. of thiourea. The solution was heated atreflux for l /2 hours. A solution of 4.5 g. of potassium hydroxide in 65ml. of water was added and the mixture was heated at reflux for anadditional l V: hours. The alcohol was evaporated at 15 mm. (32) and theaqueous residue was neutralized with concentrated hydrochloric acid topH 3.5. After cooling for 2 hours in an ice bath 3.5 g. of 2-mercapto-l3.4- thiadiazole as yellow crystals were collected adn weighed 3.5 g.M.p. 125127. The IR and NMR spectra were consistent for the structure.

7-Amino'3-( 1.3.4-thiadiazolyl )-thiomethyl ]-3- cephem-4-carhoxylicacid -[2-( To a suspension of 8. l g. (.03 mole) of7-aminocephalosporanic acid and 3.5 g. (.03 mole) Z-mercapto-1.3,4-thiadiazolc in 200 ml. of .1 M phosphate buffer (pH 6.5) was addedwith stirring 5.4 g. (.064 mole) of sodium bicarbonate. The mixture wasstirred at 55 under nitrogen and all of the solid dissolved. Thestirring was continued for 3 hours and the solution was cooled to 5 andadjusted to pH 5 with glacial acetic acid. The mixture was stored for 2hours and the product, 7-amino-3-[ 2-( l .3.4-thiadiazolyl)-thiomethyl]-3- cephem-4-carboxylic acid, was collected and weighed 9g., M. 140 (slow dccomp.) The IR and NMR spectra were consistent for thestructure.

7-Amino-3-[2-( S-hydroxymethyl-l ,3,4- thiadiazolyl )thiomethyl]-3-cephem-4-carboxylic acid COOH of 2-amino-1,3.4-

l -Hydroxyacetylthiosemicarbazide A mixture of 18.2 g. (0.2 mole) ofthiosemicarbazide and 30.4 g. (0.4 mole) of glycolic acid was heatedtogether at 90 for 2 hours with good stirring. The mixture was cooled atroom temp. and washed with cold abs. alcohol. The solid was collectedand air dried to weigh 40 g. The product was recrystallized from ml. ofboiling 50% alcohol to yield 23 g. of the crystalline1-hydroxyacetylthiosemicarbazide. M.p. l89-l90; Anal. Calcd. for C -,H NOS, C. 24.18; H, 4.74; N, 28.21; S, 21.51.

Found: C, 24.08; H. 4.50; N. 27.67; 5. 21.35. The IR and NMR spectrawere consistent for the structure.

Z-Aeetoxymethyl-S-amino-1,3,4-thiadiazole A mixture of 22.4 g. (0.15mole) of 1-hydroxyacety1 thiosemicarbazide and 60 ml. of acetyl chloridewas stirred at room temperature for 2 hours and then heated to 40 untilall the solid had dissolved (ca. 2 hours). The acetyl chloride wasremoved under reduced pressure 40 (15 min.) and the residue was stirredwith 100 ml. of ice water. The mixture was adjusted to pH 9.2 with 10%KOH and the product was collected. The crude solid was recrystallizedfrom absolute alcohol to yield after air drying 6 g. of crystalline2-acetoxymethyl-S-amino-1.3,4-thiadiazole, M.p. l9 l192 C.

Anal. Calcd. for C h N O S: C. 34.63; H, 4.07; N, 24.26.

Found: C, 35.16; H, 4.35; N. 24.38.

2-Hydroxymethyl-S-mercapto-1,3 ,4-thiadiazole The procedure of Goerdleret al. was followed using 34.6 g. grams (0.2 mole) of2-acetoxymethyl-5-amino- 1.3.4-thiadiazole and 64 grams (0.9 mole) ofsodium nitrite in 320 ml. of 48% hydrobromic acid and 0.1 gram ofpowedered copper. The bromo compound was treated with 7.6 grams (0.]mole) of thiourea and l 1.2 grams of potassium hydroxide in 25 ml. ofwater. Upon acidification with 6 N hydrochloric acid the mercaptan wasextracted in ethyl acetate and treated with 17 grams of potassiumZ-ethylhexanoate. The potassium salt was collected. washed with ethylacetate and dried to weigh 12.2 grams. The salt was recrystallized fromacetone-water to give 7.6 grams of crystalline potassium2-hydroxymethyl-5-mercapto-1,3,4-thiadiaz0le. M.p. -13]C.

Anal. Calcd. for C H KN S O'H O: C, 17.64; H, 2.46; N. 13.70.

Found: C, 17.81; H. 2.43; N. 13.66

The IR and NMR spectra were consistent for the structure.

7-Amino-3-[2-(5-hydroxymethyl-1,3,4- thiadiazolyl )thiomethyl]-3-cephem-4-carboxylic acidi The procedure followed was the same as theprocedure for the unsubstituted thiadiazole using 2.25 g. (0.012 mole)of mercaptan. 3.3 g. (0.012 mole) of 7- amino cephalosporanic acid and lg. (0.012 mole) of sodium bicarbonate in 100 ml.. 1 M phosphate bufferto yield 3.15 g. of tan, solid 7-amino-3-[2-(5- hydroxymethyll,3.4-thiadiazolyl )thiomethyl1-3- cephem-4-carboxylic acid, M.p.l70175C. decomp. The IR and NMR spectra were consistent for thestructure.

The following examples are given in illustration of, but not inlimitation of, the present invention. A11 temperatures are given indegrees Centigrade. Skellysolve B" is a petroleum ether fraction of BF.6068 C. consisting essentially of n-hexane. lR-120 is also calledAmberlite 1R-l20 and is a strong cation exchange resin containingsulfonic acid radicals. Amberlite 1R-120 is a commercially availablecation exchange resin of the polystyrene sulfonic acid type; it is thusa nuclear sulfonated polystyrene resin cross-linked with divinyl benzeneobtained by the procedure given by Kunin, Ion Exchange Resins, 2nd.Edition (1958), John Wiley and Sons, Inc. Therein see pages 84 and 87for example.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 cmNH.

S CH. .CONH [f fi rfi zSC CH N o s coon 7-[Z-Aminomethylphenylacetamido]-3-[ 2-( l,3,4- thiadiazolyl )thiomcthyl]-3-cephem-4-carboxylic acid.

A mixture of 4.8 g. (0.015 mole) of sodium 2-N-(1-carbethoxypropen-Z-yl)aminomethyl-phenyl acetate and 5 drops ofN,N-dimethylbenzylamine in 94 ml. of tetrahydrofuran at was stirredvigorously with 2. 1 g. (0.0156 mole) of isobutylchloroformate. Themixture was stirred for 5 minutes and added to a solution of 3.8 g. (.01mole) of 7-amino-3-[2-( l,3,4-thiadiazolyl)thiomethyll-3-cephem-4-carboxylic acid and 2.9 ml. ofN-methylmorpholine in 53 ml. of water at 3. The resulting solution wasstirred for 2 hours and the tetrahydrofuran was evaporated at 30 at 15mm. The solution was adjusted to pH 6.5 with cone. hydrochloric acid andstored for 3 hours at 5. The cepHalosporin was collected and weighted2.3 g., m.p. 140 decomp.

Anal. Calcd. for C,.,H ,N O S,,.H O: C, 44.64; H, 4.14; N, 13.69.

Found: C, 44.81; H, 4.31; N, 13.46.

The IR and NMR spectra were consistent for the structure.

Potassium 7- 2-N-salicylideneaminomethylphenyl )acetamido1- 3-[ 2-( 1,3,4-thiadiazolyl )thiomethyl l-3-cephem-4- carboxylic acid.

To one gram (0.02 mole) of the corresponding cephalosporanic acid fromExample 1 in 15 ml. of methanol was added 280 mg. (0.023 mole) ofsalicylaldehyde and 200 mg. (0.020 mole) of tricthylamine. The solutionwas heated to 50 on the steam-bath, filtered and 360 mg. of potassium2-ethylhexanoate was added. The methanol was evaporated at 30 15 min.)to one-half volume and the potassium salt was collected and weighed 650mg, m.p. 145 decomp.

Andv CulCd. f0! C2 H2qKN505S1fh2OI C, H, 33.79; N, 10.98. Found: C,48.99; H, 4.04; N, 10.55. The IR and NMR were consistent for thestructure.

7-( 2-Aminomethylphenylacetamido )-3-[ 2-( 5- hydroxymethyl- 1,3,4-thiadiazolyl)thiomcthyl1-3- cephem-4-carboxylic acid.

The procedure followed was the saem as the procedure used for theunsubstituted 1,3,4-thiadiazole cephalosporin in Example I. The enamine3.6 g. (0.012 mole), isobutyl chloroformate, 1.6 g. (.012 mole) and 0.4ml. of N,N-dimethylbenzylamine in 71 ml. of tetrahydrofuran at 40 wasadded to a solution of 2.9 g. (0.1 mole) of the amino-cephalosporanicacid in 2.3 ml. of N-methylmorpholine and 41 ml. of water at 0. A yieldof 1.6 g. was obtained m.p. decomp.

Anal. Calcd. for C H N O S -H O: C, 45.69: H, 4.40; N, 13.32. Found: C,45.34; H, 4.30; N, 12.9].The IR and NMR spectra were consistent forstructure.

Potassium 7-[(Z-N-Salicy1idencaminomethylphenylacetamido]-3-[Z-(S-hydroxymethyl-1,3,4-thiadiazoly1)thiomethy1]-3-cephem-4-carboxy1ic acid.

ethylhexanoate (KEH) in ethyl acetate to a solution of the cephalosporin(zwitterion) in DMSO to precipitate thedesired potassium salt.

The procedure used was the same as the procedure used for theunsubstituted thiadiazole. The 750 m g EXAMPLE 6 (0.0015 mole) ofcephalosporm from Example 3, 162 mg. (0.0016 mole) of triethylamine, 219mg. (0.0018 The sodium salt of the compound of Example 3 was mole) ofsalicylaldehyde and 292 mg. (0.0016 mole) of prepared by pre-formatlonof Its dlethylammomum salt potassium Z-ethylhexanoate in 10 m1. ofmethanol gave In methanol f0|1QWd y addition of a Solution of 0- 530 mg.of yellow crystals. M.p. 140 slow decomp. dium Z-ethylhexanoate (SEH) inethyl acetate and then Anal. Calcd. for C -,H KN,,O,,S C, 48.55; H,3.92; dilution with isopropanol to precipitate the desired so- N, 10.34.Found: C, 49.03; H, 4.48; N, 10.28. dium salt which had a highsolubility in water.

The IR and NMR spectra were consistent for the The following tableillustrates the potent in vitro acstructure. tivity of the compounds ofthe present invention and E AMP E 5 also illustrates the marked decreasein activity, particu- X L larly against Gram-negative bacteria, producedby The potassium salt of the compound of Example 1 seemingly minorvariations in the thiol attached at the was prepared by adding asolution of potassium 2- 3-position.

M.1.(. (pk/ml.)

Nutrient Broth Ex. 1 Ex. 3

Organism MR-S MRS BL-S BL-S BL-S BL-S BL-S BL-S BL-S BL-S BL-S D.pncumoniae 110" A9585 .0021 .016 .004 .008 .03 .004 .016 .008 .016 .03.0021 Str. p)llgcflt.s* 1 10 AIhtH- 0021 .016 .004 00s .03 .004 .016.003 .016 .06 .016 s. aurcus Smith 110 A9537 06 .013 .06 .013 0.13 .030.25 .06 0.13 0.25 0.13 S. aurcuH-SOJ scrum A9537 0.13 0.25 0.13 0.250.25 0.13 0.25 0.5 0.5 0.5 0.25 1 10-0 5 aurcus Bx1633 110-0 A9600 0.160.25 .014 11.16 0.3 .08 0.6 0.16 0.3 0.6 0.3 s. RILIICUS 100633 (10A9600 0.5 0.25 0.16 0.3 0.6 .011 1.3 0.16 0.3 0.6 0.6 s. auruusMet1i.-R1:s 10 1 1 115097 1 2 0.5 1 1 0.5 2 0.5 4 2 2 $111 untcrititlis1 10 *1 A953] .08 0.16 .04 0.16 0.3 0.16 0.6 0.16 0.6 0.6 0.3 F.01111.11'11110 A15119 0.3 0.6 0.3 0.3 0.6 0.6 2.5 2.5 2.5 2.5 1.3 F.LU11(111 A9675 1 2 2 1 1 2 4 4 s 4 4 K. pneumoniae 1 10') A9977 .08 0.60.161 0.10 0.6 11.3 1.3 1.3 2.5 1.3 0.6 K. pneumoniae (10 A15130 1 2 0.50.5 2 1 2 16 4 4 2 Pr. mirahilis 1 10 A9900 0.3 0.6 0.16 0.3 0.6 0.3 1.31.3 1.3 1.3 1.3 Pr. morganii 110"] A15153 2 4 4 4 1h 4 16 1h 63 b3 16Ps. ucruginosa (10") Ami-BA 125 125 125 12 125 125 125 125 125 125 125Scr. marcescuns 1 10 J A2001) 8 1h 16 1b (13 63 125 125 125 125 125 Em.CIOEICHC (10 *1 A9050 32 63 125 16 125 125 125 125 125 63 125 1261.LIUiiLflC (10 A0657 0.16 0.3 0.16 0.16 0.25 0.3 1.3 .5 1.3 1.3 0.315111. cloncm: 10 A9659 1 2 1 1 4 4 8 1 32 1b 4 *'1 Antibiotic AssayBroth 5')? serum Nutrient Broth. "Dilution of overnight broth culture.

O N -(H. .S-R ('H. .NH:,

* O0 MR-S MR-S BL-S BL-S BL-S BL-S BL-S 9-1 16 085 090 664 683 684 T l 1*1 1? 1"?1, 1"1 '1 1 1" R= )1 )EHJK H oH k 1v k )C'HQOH T1 5 s S 0 S OCH,

BL-S BL-S BLS BLS 692 693 695 696 r! 0 S I O If S BL-S685 and bl-s690have an antibacterial spectrum that is similar to that of MR S94 but areabout 2-fold less active. However, they are somewhat more soluble (2.53.5 mg./ml.) in water than MR-S94. Blood level studies show that theygive peak levels following lM administration of IO mg./kg. that arecomparable to that of MR-S94. Neither was absorbed to a significantdegree when administered orally. The percentage of these drugs bound tohuman serum proteins was 52 for BL- S685 and 36 for BL-S69O as comparedto SI for MR- S94. In rat crystalluria studies [BL-S685 and BL-S69O gavesatisfactory high serum and urine concentrations without any evidence ofcrystalluria.

We claim:

1. The acid having the formula the acid of claim 1.

1. THE ACID HAVING THE FORMULA
 2. The sodium salt of the acid ofclaim
 1. 3. The potassium salt of the acid of claim
 1. 4. The zwitterionform of the acid of claim
 1. 5. A nontoxic, pharmaceutically acceptablesalt of the acid of claim 1.